The present application relates generally to medical devices and methods and more particularly to microfluidic devices and methods for processing cells and molecules for therapeutics (stem cell therapy, gene therapy) and diagnostics (e.g., characterizing, amplification, sensing, processing, enriching circulating tumor cells.) using different biospecies (e.g., different cells, cells containing different substances, different particles, different biochemical compositions, genes, proteins, enzymes etc.).
‘On-the-Fly Field-Potential Sensing Electrode Track’ Technology for Stem Cell Sorting
The derivation of patient-specific reprogrammed somatic cells makes immunologically compatible stem cell replacement strategy very attractive for several applications such as spinal cord therapy. Pluripotency has been derived at increased efficiencies from several easily accessible human cell types, including blood cells, keratinocytes and dermal fibroblasts enable drug screening, disease modeling and autologous cell transplantation in clinical therapy. The major challenge in this tissue replacement therapy is the establishment of effective isolation of differentiated cells to avoid teratoma formation. However, at present, it is unclear whether any of the currently available strategies to generate differentiated cells from iPSCs is able to eliminate the risk of teratoma formation. Moreover, the current separation method—microscope-assisted manual isolation—is error-prone, time-consuming, and labor-intensive, and does have the capacity to sort multiple cell phenotypes. Fluorescence-activated cell sorting is capable of sorting multiple phenotypes but uses labeling of cells. Magnetic-activated cell sorting also lacks the robustness to sort out more than one cell type at a time. Unfortunately, conventional separation techniques requiring exogenous labeling or genetic modification is not suitable for clinical applications and so superior iPSC sorting is an urgent unmet need. The novel flow based high throughput label-free sorting scheme to separate stem cells and their differentiated progeny for regenerative medicine using cell sorting based on their response to electrical stimulation called ‘On-the-Fly Field-potential Sensing Electrode Track’ (OFFSET) technology is significant.
This high throughput integrated OFFSET platform can be configured to:                precisely flow and focus high throughput single-cells        accurately detect single cells using impedance sensing        stimulate the detected cells triggered by the impedance sensor        detect their stimulated response through an electrode array        process the stimulus response signature in real time        perform fluidic switching with the comparison of pre-stored stimulus response signatures        
In microfluidics, surface electrodes have been used to detect electrical signals from cells such as extracellular ionic currents producing a characteristic field potential (FP) signal in different ion channels and gating kinetics. Though these electrophysiological measurements have been used to identify subpopulations of electrically-excitable cells, at static conditions, high throughput flow based ‘on the fly’ recording have not done so far which are very essential for clinical applications. These FP signals can be monitored during stem cell differentiation and are characterized as a marker for endpoint analysis of embryonic stem cells in order to quantify ion channel expression levels and cell maturity or phenotype. Our innovation is to develop a flow based system to sense the degree to which stem cells have differentiated into these cell types using ‘On-the-Fly Field-potential Sensing Electrode Track’ (OFFSET) technology. Therefore OFFSET technology offers a label-free cell sorting of stem cells and their differentiated progeny based on their response to electrical stimulation with several advantages. In potential therapeutic applications, the cell populations relevant for therapy can be electrically excited and the resulting transmembrane ion currents are measured using an array of surface microelectrodes along the direction of the flow. The electrical current measurements in response to electrical stimulation for differentiated states of the cells are built up as electrical signatures for real time comparison and sorting. Since these transmembrane ion currents are measured non-invasively to sort the differentiated cells based on these field potential markers, the sorted cells are highly viable for therapeutic applications. Therefore ventricular-like cardiomyocytes from iPSC derived populations can be separated for cardiac tissue replacement therapies in an ‘on the fly’ system at high throughput scale. These cell populations for such sorting include cardiomyocytes, neurons, skeletal muscle, and vascular smooth muscle. The OFFSET platform combines the technologies of flow based field potential sensing, high speed signal processing and high throughput microfluidic cell sorting to rapidly detect, identify, and sort millions of specific cells for downstream applications. Our endeavor to overcome the barriers that prevent successful translation of stem cell biology into clinical therapy is highly significant to improve human health and control of human diseases.
Flow Driven Blood Based Sorting of Cancer Cells Using Multi Spiral Fluidic Channels
Sorting of cancer cells particularly circulating tumor cells (CTCs) from blood is important for clinical diagnostics. Despite the progress in early diagnosis and introduction of new therapy regimes, cancer remains a prominent health concern in modern societies with one in four deaths in the US and a total of 1,529,560 new cancer cases with 569,490 deaths from cancer projected in 2010. The early dissemination of the cancer and the systemic spread of tumor cells to other parts of the body results in a negative prognosis and death. Such CTCs can be found in the peripheral blood of patients before the primary tumor is detected. Therefore CTCs are fluid biopsy for primary tumor cells sampled as a minimally invasive, prognostic and predictive marker to reflect the biological characteristics of tumors and are implemented in an increasing number of clinical studies. These CTCs play a pivotal role in changing the biology and marker expression compared with the primary tumor and so detection and characterization of these cells are believed to have a substantial clinical impact on the prognosis and optimal disease management of cancer patients. In addition to a potential role in early diagnosis and prognosis, the detection of CTCs can guide therapeutic strategies for personalized treatment of patients with metastatic cancer. Identification, enumeration and characterization of CTC through immunocytochemistry, fluorescence in situ hybridization assays and all relevant molecular techniques. However, the most challenging obstacle in the separation and detection of CTCs is their extremely low concentration. Due to the rarity of the CTCs, existing immunomagnetic cell separation techniques lack the ability to separate all types of CTCs directly from whole blood at rapid and low cost.
Quantification of CTCs through the use of magnetic bead-conjugated antibodies against epithelial-cell adhesion molecule (EpCAM) remains a point of discussion for treatment decisions. EpCAM-dependent assays are based upon the assumption that the presence of epithelial cells in peripheral blood indicates the presence of tumor cells. However, epithelial cells may be found in healthy donors and EpCAM-based assays are not able to detect normal-like tumor cells. Moreover, certain tumor types such as melanoma are not of epithelial origin which suggests that EpCAM-based assays may be of limited use. Epithelial antigen may be lost on CTCs due to the epithelial-mesenchymal transition (EMT), which is considered to be a crucial event in the metastatic process. Furthermore, CTCs must be isolated alive for testing their potential capacity to initiate tumor formation in animal models and must become easily accessible to a large range of molecular biological analysis. Therefore flow driven blood based inexpensive on-chip high performance sorting using yoked channels (f-BIOPSY) system that can isolate, quantify, and analyze circulating tumor cells from a blood sample under inertial fluidics conditions using successive approximation sorting method is of great significance. The f-BIOPSY system as compared in Table 1 can prepare CTCs for analyzing relevant cellular and molecular biological techniques for potential genetic abnormalities without using an antibody based assay. The f-BIOPSY system could have a profound influence on the early diagnosis, prognosis, early detection of relapse, and the development of new targeted strategies.
TABLE 1Comparison of f-BIOPSY technologyVeridexXanapathBiopicoCharacteristics(Cell Search)(I-SCOPE)(f-BIOPSY)ThroughputLowMediumHighSpecificityLowLowMediumIntegrationLowLowHighFluid handlingRequiredRequiredNot requiredSorting/EnrichmentImmuno-Immuno-Inertial fluidicsmagneticmagneticMultiplexityLowLowHighCell viabilityLowLowHigh
In the f-BIOPSY device, blood flow passes through a “yoked” spiral channel using successive approximation method, allowing size-selective isolation of rare tumor cells under fully reproducible and standardized inertial fluidics conditions. The f-BIOPSY device is a low-cost innovative technology with the aim of achieving isolation of tumor cells without the requirement for large and expensive apparatus and is compared with other leading systems in Table 1. Compared previous spiral method, the clinically usable highly innovative f-BIOPSY system has several advantages as characterized in Table 2. The cells isolated from the device are can be analyzed using all relevant cellular and molecular biological techniques pertinent to the identification and characterization of CTCs and their potential genetic abnormalities. This label free system can isolate living cells, allowing further tissue culture experiments. The goal of this cell sorting system is to avoid harming the cells during sorting so that high performance multianalyte detection using mRNA expression profiling can be achieved. Furthermore, tumor cells can be isolated without using an epithelial antibody-based assay, suggesting that the f-BIOPSY device can be used for the isolation of a large spectrum of tumor cells, including cells of non-epithelial origin. They can be used within clinical trials as a basis for early therapy stratification and monitoring to replace expensive and adverse radiological imaging techniques. The f-BIOPSY technology can be sensitive and reliable to allow detection and analysis of CTCs routinely for the early diagnosis, risk stratification in the adjuvant setting, early detection of relapse, the development of new targeted strategies and guiding treatment decisions.
TABLE 2Innovation of f-BIOPSY SystemSpecificationPrevious Methodf-BIOPSY SystemSpiralSingle spiralDouble spiral or multiplespiralsWidthConstantDecreasing on one spiral andincreasing on otherMainSimple channelExpansion structures in mainchannelchannelSampleRBC and leukocytesContinuously RBC and otherconcentrationare sorted out atcells are extracted out inthe end the channelby-pass channels from onespiral to another spiralchannelsSortingInefficientEfficient due to increasingefficiencychannel widthMultianalyteNot accurateCascaded branched spiralsortingchannels with defined geometryenable multianalyte sortingRapid sorterconstant width makesLarger effective width ofslow sortingparallel spiral channelsenables high flow rate sorting.Serum Based Mobile Driven Analyzer for Rapid Tests
The advent of personalized diagnostic approaches demands highly flexible analytical devices to perform multiplexed analysis for determining a wide range of disease biomarkers such as enzymes, antigens and nucleic acids and therapeutic agents. In this regard, lab-on-chip microfluidic devices paved the way for miniaturized, self-standing analytical systems and technological solutions for integration, multiplexing and programming of such biospecific reactions. Panel assays with “ad hoc” biomarkers for monitoring the health status and the drug efficacy in a specific patient is an urgent need for these devices. With this respect, these devices could be operated by a patient, a nurse, a physician or a technician directly in the doctor's office or at the patient's bed to rapidly provide all the clinical chemistry information necessary for accurate diagnosis and patient follow-up. With the increasing power of computation, communication and versatility of smart phones, in recent times, it is useful to configure the smart phones with fluidic devices for point of care diagnostics. However, performing such rapid blood based diagnostics would require development of appropriate technologies for preprocessing blood and measurement for personalized assays. Therefore, Serum based Mobile driven Analyzer for Rapid Tests (SMART) is of great significance. This effort can separate serum from whole blood, to perform diagnostics and to interface with smart phone to tap its power through cloud computing and making the diagnostics information available in private networks for its better utilization. In this system, personal diagnostics information from whole blood is derived using a microfluidic chip and transmitted to cloud computing network for access to relevant users. The innovations are as follows:                1. The SMART system can utilize a rapid serum sorting scheme for colorimetric diagnostics of test panels from serum.        2. Rapid pumping of serum in the SMART system is accomplished by acoustic steaming driven integrated passive pump.        3. Colorimetric measurement imaging is accomplished by integrated lensless optics so that compact smart phone camera can be utilized for quantification.        4. Computation, analysis and communication of the diagnostics data to a cloud computing system is enabled by the smart phone for remote multiuser access.        
The innovations in SMART system combines the technologies of serum separation in spiral fluidic channel, acoustic cavitations streaming pump, colorimetric assay for test panels, smart phone based imaging and health information communication through cloud computing to develop a powerful platform for space exploration related point of care diagnostics and commercialization. The system could carry out in a quick, multiplex diagnostics in a cost-effective fashion for crew health monitoring and clinical diagnostic or therapeutic purposes. As an example, simultaneous quantitative analysis of glucose, lactate, and uric acid in blood samples is applied.
The device allows short analysis time due to miniaturization and high flexibility of assay and format, as well as a potential costs reduction. Its key advantage is that the different types of assays described above can be performed simultaneously exploiting microarray configurations. This multiplexing capability can permit the development of chips for the detection of panels of diagnostic biomarkers, e.g., for performing the diagnosis of an infectious disease by detecting both the pathogen nucleic acids and the host antibodies produced in response to the infection or to assess the liver function by combining routine clinical chemistry analyses (bilirubin, cholesterol) with the measurement of serum enzyme activities (alkaline phosphatase, aspartate, and alanine aminotransferase) and the detection of a present or past hepatitis viral infection. Thanks to the flexibility of chip fabrication, chips to evaluate panels of biomarkers could be also specifically developed for a given patient to perform “personalized medicine” on the basis of genetic approaches.
Parallel Incubators with Loaded Single Cells for Lysis and Amplification Reactions
While the cell is recognized as a fundamental unit that can generate a complex organism containing cells with diverse patterns of gene expression, only a limited number of measurement techniques permit single cell resolution that is interesting to the biological, medical, and pharmaceutical communities. Traditional methods of gene expression analysis examine pooled mRNA from thousands of cells, resulting in an averaged picture of gene expression across an entire cell population. This restricts the ability to distinguish between the individual responses inherent cell-cell heterogeneity within a sample and to disentangle the complexity of the regulatory mechanisms controlling specific responses. The primary problems that hinder such single cell analyses are difficulty in handling a minute amount of sample, inability to prepare and manipulate single cells, inability to have high-throughput capability, and not being able to integrate with amplification protocols and detection mechanisms. Further, the application of current single cell PCR techniques is limited by long turnaround times, high cost, labor intensiveness, the need for special technical skills, and/or the high risk of amplicon contamination. The need for single-cell mRNA analysis is evident given the vast cellular heterogeneity of all tissue cells and the recent developments in whole genome amplification procedures, single-cell complementary DNA arrays and single-cell comparative genomic hybridization. When genetic analysis of single cells becomes a common practice, new possibilities for diagnosis and research can open up. Current attempts to perform high-throughput single cell dispensing and analysis involves flow cytometry and robotics. However, such systems are expensive as a routine research or diagnostic device. Therefore, Biopico Systems, Irvine identified this opportunity to develop a ‘Parallel Incubators with Loaded single cells for Lysis and Amplification Reactions (PILLAR)’ system to perform high-throughput quantitative single-cell gene expression profiling. This system can provide unique information critical not only to the quality control and clinical translatability of iPSCs for regenerative medicine but also to understand the relationship between transcriptional and phenotypic variation in the development of pathology, oncogenesis, and other processes of a target cell. For example, precise molecular analysis of abnormal gene and proteins in single cells from a large population of cells helps in cell clonality, genetic anticipation, single-cell DNA polymorphisms and early diagnosis of cancer, infectious diseases and prenatal screening.
PILLAR technology, first traps single cells at configured micropillars and then encapsulates using immiscible fluidics around the single cell traps as picoliter reservoirs. The novelties pertaining to the development of the PILLAR system include 1) trapping of single cells with a set of micropillars (and guiding pillars for focusing the cells towards traps) 2) formation of microreactors using immiscible fluids around a single cell and 3) thermocyling of these anchored microreactors using flow of oil at two different temperatures for fast and reliable PCR reaction. The all-in-one PILLAR system combines commercially available fluorescent RT-PCR with immiscible microfluidics and single cell microtrapping technologies for performing thousands of single cell RT PCR in picoliter volumes in a quick, high-throughput, and cost-effective fashion. The PILLAR system can help to understand the relationship between stochastic variations of gene expression within individual cells and heterogeneous transcriptional profiles across a population of cells.
This highly integrated PILLAR platform is configured:                to precisely trap high throughput single-cells in array of micropillar based trapping sites        to encapsulate single cells as picoliter reactors by flow of immiscible fluids        to rapid thermal cycling on anchored single droplets at the micropillar trapping sites        to perform thousands of single-cell PCR for regenerative medicine or clinical diagnosticsThis PILLAR platform would be very useful for accurately quantifying the differentiation process and could serve as a performance metric of every step of stem cell differentiation process for regenerative medicine. The combination of high-fidelity manipulation of single cells and the ability to perform nucleic acid amplification offers the possibility of developing a powerful automated instrument which has highly significant commercial applications such as cancer diagnostics and prenatal diagnostics.        
Biochemical analysis of genes and proteins from single cells is of significant interest. The intellectual merit of the technology is to develop a chip/system that entraps single cells and performs 10,000 parallel single cell RT-PCRs simultaneously in a cost-effective fashion. This can provide a means to perform precise molecular analyses on single cells from large populations of cells using a highly sensitive approach. The chip combines three unique microfluidic techniques for the automation of a cell-based real-time PCR-based diagnostic system on a chip with supporting analytical instrumentation. This novel device can differentiate gene expression of a particular rare cell from other single cells with a capability of multiplexing for the detection of house-keeping genes and target genes. This capacity to analyze large populations of individual cells could provide unique opportunities in the life sciences and support biomedical research activities in the fields of virology, oncology and pathology.
The broader/commercial impact of the project is the development of a cost-effective solution for diagnosis of various diseases using cellular gene expression variation of multiple single cells for leukemia, prenatal screening, genetic screening of multiple diseases, or the detection of viruses such as HIV virus. Isolation of single cell in a high throughput fashion followed by gene expression analysis can lead to several research findings. Such findings can lead to early detection and prognosis of diseased states including differential detection of an infected cell from uninfected cells, detection of cancer markers in different cells, and changes in gene expression of diseased single cells at high speed and high specificity. Further, small concentration changes and/or altered modification patterns of disease-relevant components, such as mRNA and/or micro RNA, have the potential to serve as indications of the onset, stage, and response to therapy of several diseases. In current, manual, single-cell PCR methods low abundance mRNA is often lost during cell lysis and extraction processes and these methods are also extremely labor intensive requiring expensive equipment to isolate single-cells to perform PCR. The PILLAR system can detect rare abnormal cells and carry out single-cell PCR or RT-PCR. Micrototal analysis systems of the prior art have typically involves microdroplets formation followed by cells and molecules encapsulation. These technologies require external active instruments to accomplish medical diagnostics which can be difficult to perform one touch or one step device operation. On the other hand, in our technology, single or multiple cells or molecules are trapped first either at configured micropillars or nozzles and then picoliter volume reservoirs or reactors are formed around the single cell using immiscible fluidics. The isolated single cell or cells or molecules are processed for multistep temperature reactions or chemical reaction. Multiple temperature biochemical reaction such as PCR or linear isothermal amplification can be carried out on the trapped picoreactors by flowing immiscible fluids such as oil around the picoreactors. Further, if needed, a slip and lock chip technique supplies additional reagent from another layer of microfluidic chip. PCR using fluid flow thermal cycling is also highly innovative. The dimensions of the pillars for trapping cells and flow rate of the oil for encapsulating single cells are optimized for the efficiency and specificity of the diagnostics device.
Programmable Array of Living Cells for Combinatorial Drug Screening
Combinatorial drug screening for cell based drug discovery and efficacy is increasingly dependent on high-throughput technologies due to the need for more efficient screening of multiple combinatorial drug candidates. Massively parallel analytical screening technologies are needed for the exploitation of biological insight in the oncology clinic since cellular responses to anti-cancer modalities have been stochastic in nature. Miniaturized reactors have been developed to reduce culture volume, increase process efficiency and to administer chemotherapeutic drugs sequentially or together in combination for massive experimental parallelization of real-time drug screening routines. The use of combination therapies can lead to increased efficacies at significantly lower doses and side-effects and so investigation of combination therapies for curative and palliative care is very significant. Therefore an automatic Programmable Array of Living cells (PAL) that integrates on-chip generation of drug concentrations and pair-wise combinations with parallel culture of cells is significant for drug candidate screening applications.